Synthesis, Photophysical Properties, and Biological Evaluation of

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Synthesis, Photophysical Properties, and Biological Evaluation of TransBisthioglycosylated Tetrakis(fluorophenyl)chlorin for Photodynamic Therapy Shiho Hirohara, Chio Oka, Masayasu Totani, Makoto Obata, Junpei Yuasa, Hiromu Ito, Masato Tamura, Hirofumi Matsui, Kiyomi Kakiuchi, Tsuyoshi Kawai, Masashi Kawaichi, and Masao Tanihara J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01262 • Publication Date (Web): 12 Oct 2015 Downloaded from http://pubs.acs.org on October 13, 2015

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Journal of Medicinal Chemistry

Synthesis, Photophysical Properties, and Biological Evaluation of Trans-Bisthioglycosylated Tetrakis(fluorophenyl)chlorin for Photodynamic Therapy Shiho Hirohara a,*, Chio Oka b, Masayasu Totani c, Makoto Obata d, Junpei Yuasa c, Hiromu Ito e

, Masato Tamura e, Hirofumi Matsui e, Kiyomi Kakiuchi c, Tsuyoshi Kawai c, Masashi Kawaichi

b

a

, Masao Tanihara c Department of Chemical and Biological Engineering, Ube National Collage of Technology, 2-

14-1 Tokiwadai, Ube 755-8555, Japan b

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama

8916-5, Ikoma, Nara 630-0192, Japan c

Graduate School of Materials Science, Nara Institute of Science and Technology, Takayama

8916-5, Ikoma, Nara 630-0192, Japan d

Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi,

Kofu 400-8510, Japan e

Faculty of Medicine, University of Tsukuba, 1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8573,

Japan

ACS Paragon Plus Environment

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ABSTRACT: Trans-bisthioglycosylated tetrakis(fluorophenyl)chlorin (7) was designed as a powerful photodynamic therapy (PDT) photosensitizer based on the findings of our systematic studies. We show here that trans-bisthioglycosylated structure of 7 enhanced its uptake by HeLa cells and that the chlorin ring of 7 increased the efficiency of reactive oxygen species generation under the standard condition of our photocytotoxicity test. The versatility of 7 in PDT treatment was established using weakly metastatic B16F1 melanoma cells, metastatic 4T1 breast cancer cells, the RGK-1 gastric carcinoma mucosal cell line, and three human glioblastoma cell lines (U87, U251 and T98G). The pharmacokinetics of 7 in mice bearing 4T1 breast cancer cells showed a high tumor-to-skin concentration ratio (approximately 60) at 24 h after intraperitoneal injection. The PDT efficacy of 7 in vivo was approximately 250-times higher than that of monoL-aspartyl

chlorin e6 (9) in mice bearing 4T1 breast cancer cells.


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INTRODUCTION

Since the discovery of hematoporphyrin derivatives (HPD) by Lipson and Baldes in 1960, photodynamic therapy (PDT) and photodynamic diagnosis have developed via interdisciplinary studies, including medicinal science, chemistry, physics and engineering.1,2 The design and synthesis of novel photosensitizers are central to the development of efficient PDT modalities, particularly reduction of the doses of drug and photoirradiation by increasing photocytotoxicity, selective accumulation in the tumor, and rapid clearance after treatment to reduce side effects such as prolonged skin sensitivity.3 The semi-purified HPD is one of the first generation of photosensitizers approved in many countries; selectivity and photocytotoxicity of these agents are insufficient for cancer therapy. Further, first-generation photosensitizers require relatively shorter wavelengths of light, which are less penetrable to tissues. Therefore, much effort has been devoted to develop second-generation photosensitizers,4 which exert sufficient photocytotoxicity

using

light

at

wavelengths

>650

nm.

5,10,15,20-Tetrakis(m-

hydroxyphenyl)chlorin (m-THPC), benzoporphyrin derivatives monoacid ring A (verteporfin), and mono-L-aspartyl chlorin e6 (9) (Chart 1) are representative second-generation photosensitizers in clinical use. Nowadays, the photosensitizers having a high selectivity for accumulating in tumor and an additional function have been extensively studied to develop thirdgeneration photosensitizers.4 Although naturally occurring and synthetic tetrapyrrole macrocycles, such as porphyrins, accumulate in a tumor site, other mechanisms for tumor accumulation, such as active-targeting using tumor-homing elements or passive-targeting using enhanced permeation and retention effects, are keys to the development of third-generation photosensitizers. Photosensitizers linked to an antibody,5-7 peptide,8-13 folate,14-17 and a carbohydrate were synthesized, and their


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photodynamic effects were investigated. The conjugation of photosensitizers to an antibody against a tumor-associated antigen is a straightforward approach for this purpose.

Many

antibody-drug conjugates, such as rituximab and trastuzumab were developed for cancer therapy. For example, Bryden et al. reported the synthesis of zinc porphyrin derivatives bearing F(ab’)2 fragments through sequential digestion of trastuzumab, which is a monoclonal IgG1 antibody against the HER2 receptor, and demonstrated excellent photocytotoxicity for HER2-positive cell lines (BT-474 cells).5 This is the most efficient approach for drug delivery to the tumor region and the macromolecular structure reduces skin sensitivity. However, this approach is expensive and frequently presents problems in the preparation of the conjugate due to the hydrophobicity of photosensitizers and the relatively large size of an antibody. Certain oligopeptides comprising three to ten amino acid residues can act as homing elements to a specific tissue and are called cell-targeting peptides (CTPs).18 Among the CTPs, the ArgGly-Asp (RGD) peptide is the most extensively studied. The RGD sequence binds the integrin αvβ3 receptor, which plays an important role in angiogenesis associated with solid tumors. Srivatsan et al. synthesized 2-(1’-hexyloxyethyl)-2-devinylpyropheophorbide-a (HPPH) bearing a cyclic RGD peptide and demonstrated its enhanced photodynamic effect in mice implanted with integrin αvβ3 positive 4T1 breast cancer cells.10 Recently, the 14-mer bombesin, which is a high-affinity ligand for gastrin-releasing peptide receptor, was conjugated to a photosensitizer for use in PDT.8 CTPs are more cost-effective ligands than antibodies, and it is relatively easy to prepare uniform conjugates. However, peptides frequently induce cytotoxicity and are costly to mass-produce. Folate is a frequently used small molecule for targeting tumors because the folate receptor (FR)-α isoform is overexpressed by many types of tumor cells.

Gravier et al. synthesized a m-THPC-like


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photosensitizer conjugated to folate via an ethylene glycol linker and demonstrated its enhanced accumulation in mice xenografted with FR-s-positive KB cells.17

Therefore, folate is a

promising ligand for tumor targeting; however, the highly hydrophobic nature of folate molecules frequently brings a synthetic problem as well the significant heterogeneity of their pharmacokinetics in vivo. Carbohydrates are essential molecules for life as an energy source and as ligands that mediate important biological functions.

The enhanced uptake of glucose by malignant cells is an

established strategy for cancer diagnosis and a promising approach for targeting therapeutics to tumors.

Therefore, numerous photosensitizers linked to carbohydrates were developed and

tested for their efficacy for PDT.19-21 During the past decade, we synthesized a number of glycosylated tetraphenylporphyrin and chlorin derivatives for use as PDT photosensitizers. The drug concentration inducing 50% cell death (EC50 value) of most simple glycoconjugated tetraphenylporphyrin22 (Chart 2) prepared in our laboratory was 5 µM for HeLa cells under certain conditions.

From the starting photosensitizer, we synthesized 57 glycoconjugated

photosensitizers with the following structural variations: (1) type of carbohydrate and oligosaccharide22-26, (2) position of glycosylation at a meso-phenyl group (para- or meta-)22-25, (3) photoabsorbing functionality (porphyrin or chlorin)23-26,29, (4) peripheral phenyl ring (phenyl or tetrafluorophenyl groups)25-30, (5) heavy atom effect of the central metal ion27,29,30 and (6) the substitution pattern of glycosylation28,30. All glycoconjugated photosensitizers were tested for their photocytotoxicity under the same conditions.

Finally we found that trans-

bisthioglycosylated tetrakis(fluorophenyl)chlorin 7 (Chart 2) is the PDT photosensitizer with the highest photocytotoxicity (EC50 of approximately 0.001 µM reported in this article under the same conditions). In the present study, we report the synthesis and photochemical properties of


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7, including its photocytotoxicity to eight cell lines derived from cancers of the cervix, skin, breast, stomach, and brain as well as the in vivo pharmacokinetics and photodynamic effect in mice bearing 4T1 breast cancer cells. RESULTS AND DISCUSSION Synthesis and Characterization. The approaches to prepare 7, were as follows: (1) transbisthioglycosylation of the corresponding chlorin and (2) reduction of the corresponding transbisthioglycosylated porphyrin. Because the first approach formed a large amount of by-products, we adopted the second approach. The precursor 1 (Scheme 1) was prepared from 5,10,15,20tetrakis(pentafluorophenyl)porphyrin (TFPP) and acetyl 2,3,4,6-tetra-O-acetyl-1-thio-β-Dglucopyranoside, according to our published methods.28 Following chromatographic separation of 1, the trans-substitution was confirmed using 1H NMR spectroscopy.

The 1,3-dipolar

cycloaddition of azomethine ylide to yield 1 was performed in the presence of N-methylglycine and paraformaldehyde dissolved in toluene.

The purification of the crude product by

chromatography afforded trans-bisthioglycosylated chlorin 5 as dark green solids in 40% yield. Because the 1,3-dipolar cycloaddition to the β-pyrrole position breaks the C2 symmetry of 1, two diastereomers of 5 should be formed. However, the separation of the diastereomers was virtually impossible, and we therefore, used 5 as a diastereomeric mixture. The acetyl group of 5 was removed using sodium methoxide, and the crude product was purified with using an octadecylsilyl-bound silica gel column chromatography with a mobile phase containing a mixture of acetonitrile and water (8/2, v/v). The yield of 7 was 55%. The compounds 5 and 7 were characterized using 1H, 13C and

19

F NMR, UV-vis spectroscopy, electron-spray ionization

time-of-flight (ESI-TOF) mass spectroscopy, HPLC and elemental analysis.


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Figure 1 shows the UV-vis spectra of 7, 3, 8, and mono-L-aspartyl chlorin e6 dissolved in Dulbecco’s phosphate-buffered saline (PBS) containing 1% DMSO, and Table 1 lists the spectral data. The Soret bands of 7 and 3 were significantly broadened due to their poor water-solubility, whereas those of 8 and mono-L-aspartyl chlorin e6 were relatively sharp.

The maximum

absorption wavelength (λmax) of 7 was red-shifted approximately 15 nm compared with that of 8. This red-shift was also found in the porphyrin derivatives 3 and 4.28 Photosensitizers 7, 8 and mono-L-aspartyl chlorin e6 absorbed significantly over the range of 630‒700 nm, which is characteristic of the chlorin ring and more advantageous for porphyrin-based photodynamic therapy. Table 2 lists the oscillator strengths >500 nm (f>500nm) and >600 nm (f>600nm). There were no significant differences between the f>500nm values of 7, 3 and 8. However, the f>600nm values of 7 and 8 were approximately 3 or 4 times higher than that of 3. Reactive oxygen species (ROS) are key cytotoxic intermediates of photodynamic therapy. Therefore, the efficiency of the ROS generation upon photoirradiation is an important measure of the cytotoxicity of photosensitizers. The quantum yield of singlet oxygen (1O2) (Φ∆) generated by photosensitization was determined from the luminescence emitted by

1

O2, using

tetraphenylporphyrin tetrasulfonic acid (TPPS) as a standard. The Φ∆ values are listed in Table 2. The Φ∆ value of 7 was approximately 1.5-times higher than those of 3 and 8. For an electronically excited dye with a high oxidation potential, oxidation and reduction of substrates generate of ROS such as hydrogen peroxide and hydroxyl radical. The relative quantum yield of hydrogen peroxide (ϕH2O2) from 7 was much lower than those of 3 and 8, and the relative quantum yield of the hydroxyl radical (ϕ·OH) of 7 was comparable to that of 8 and much lower compared with that of 3. Note that these ROS should not be generated by the direct electron transfer from electronically excited photosensitizers to an oxygen molecule, namely a type I


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photoreaction. A likely mechanism is as follows: The energy levels of occupied orbitals of fluorinated porphyrins, such as TFPP, are very low because of the strong electron-withdrawing effect of fluorine atoms. Therefore, the oxidation potential of the electronically excited state is high.

Upon one electron reduction of the electronically excited species by surrounding

substrates, the anionic radical species generated could reduce molecular oxygen to generate superoxide and other ROS. The lower ϕH2O2 and ϕ•OH values of 7 than those of 3 may be attributed to the higher electron energy of the chlorin ring than that of the porphyrin ring. The reason for the difference in the ϕH2O2 values between 7 and 8 is under investigation. In Vitro Photocytotoxicity. The photocytotoxic effects of 7, 3 and 8 on HeLa cells were assessed using a light dose of 16 J·cm−2 at wavelengths >500 nm. Dark cytotoxicity of these photosensitizers was evaluated in HeLa cells at the photosensitizer concentration of 0.5 µM without photoirradiation. All photosensitizers showed no cytotoxicity in the dark (data not shown).

Figure 2a shows the cell survival ratio as a function of the concentrations of

photosensitizers. The EC50 value of 7 was 1–5 nM which was almost same to that of 3 (1–5 nM) and lower than that of 8 (50–100 nM). glycosylated

porphyrins,28,30

Similar to the photocytotoxicities of a series of

trans-bisthioglycosylated

chlorin

7

exerted

greater

photocytotoxicity than that of fully glycosylated chlorin 8. The superiority of 7 compared with 3 was significant at wavelengths >600 nm (Figure 2b).

Under such conditions, the

photocytotoxicity of 7 (EC50 = 0.5–1 nM) was significantly greater than that of 3 (EC50 = 10–50 nM). Because the transparency of tissue to light increases at wavelengths ranging from 500 nm to 700 nm, the light availability of 7 was more effective than that of 3. Figure 3 shows the relative uptake amount of 7, 3 and 8 into HeLa cells. The uptake amount of 7 was almost the same as that of 3 and approximately two-fold higher than that of 8. The


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enhanced uptake of trans-bisthioglycosylated chlorin 7 compared with that of fully-glycosylated chlorin 8 is similar to that of the glycosylated porphyrin series.28,30 Therefore, cellular uptake was controlled solely by the peripheral glycosylation pattern and not by the core structure. In contrast, the absorption of light was mainly due to the core structure. For the glycosylated tetrapyrolle macrocycle, therefore, its cellular uptake and photoabsorbing properties can be designed separately. Because there was no difference in the uptake amount of between 7 and 3, their photosensitizing effects should account for their cytotoxicities. Further, it is likely that the greater f>500nm and Φ∆ values of 7 impart higher efficacy than that of 3. Although the ϕ•OH value of 3 was significantly greater than that of 7, the hydroxyl radical does not seem to be a dominant cytotoxic species because the half-life of the hydroxyl radical has a critical disadvantage of a much shorter lifetime than that of singlet oxygen. Figure 4 shows the luminescence image of HeLa cells co-incubated with 7 and MitoTracker Green FM. The intense red fluorescence emitted by 7 was observed near, but not within, the nucleus (Figure 4a), and which the pattern resembled that of the fluorescence emitted by MitoTracker Green FM (Figure 4b).

The merged image indicates that 7 was localized to

mitochondria (Figure 4c), as trans-bisthioglycosylated porphyrin 3.30

Therefore, 7 is a

photosensitizers with greater cellular uptake and higher photosensitizing ability because of transbisthioglycosylation and chlorin-ring architecture, respectively. Under certain conditions of our photocytotoxicity test, 7 exerted greater photocytotoxicity than that of mono-L-aspartyl chlorin e6, which is a second-generation photosensitizer approved in 2004 by the Japanese government to treat early-stage lung cancer. Further, PDT is applied to cancers of the skin, uterus, and breast and was approved recently as a treatment for early-stage stomach cancer and brain tumor. To confirm the versatility of 7 for


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killing cell lines derived from the tumors described above, photosensitizers 3, 7, and mono-Laspartyl chlorin e6 were used to treat the weakly metastatic B16F1 melanoma cell line, the metastatic 4T1 breast cancer cell line, the RGK-1 gastric carcinoma mucosal cell line, and three human glioblastoma cell lines (U87, U251, and T98G), which are listed in order of increasing malignancy. A halogen lamp (100-W) equipped with an R-60 cutoff filter (λ > 600 nm) was used to irradiate these cells at light dose of 16 J·cm−2. The plots of the cell survival ratio as a function of the drug concentration are included in Supporting Information, and the EC50 values of 3 and 7 are listed in Table 3. The EC50 values of 7 ranged from 0.1 to 10 nM and were much lower than those of 3 for all cell lines. Therefore, 7 was highly cytotoxic to cell lines derived from diverse tumor types including highly malignant brain tumor. Tissue distribution.

The pharmacokinetics of 7 and mono-L-aspartyl chlorin e6 were

investigated using female BALB/c mice bearing 4T1 breast cancer cells. Figure 5 shows the concentration of the photosensitizers in plasma from 1 to 24 h after an intraperitoneal injection of 7 (31.25 nmol·kg−1, 0.045 mg·kg−1) or 4 h after administration of mono-L-aspartyl chlorin e6 (6250 nmol·kg−1, 5.0 mg·kg−1). Figure 5 indicates that 7 was drained from blood in 24 h like that of mono-L-aspartyl chlorin e6. These data are consistent with the findings of Ferrario et al. who reported that mono-L-aspartyl chlorin e6 administered to C3H/HeJ mice implanted with the BA mammary carcinoma was not detected in the bloodstream within 24 h.31 The concentration of photosensitizers in each tissue was quantified using a fluorometry, and listed in Table 4. Photosensitizer 7 was present in high levels in the liver, bladder, small intestine, and spleen but did not accumulate in the brain. These findings were similar to those of mono-L-aspartyl chlorin e6,32 porfimer sodium,33 sulfonated gallium phthalocyanines34, and verteporfin.35 We show here that 6 h after administration, 7 accumulated in the skin and muscle


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as well as in the tumor as did mono-L-aspartyl chlorin e6. Moreover, the tumor-to-skin uptake ratio of 7 was very high (approximately 60), 24 h after injection (Table 4 and Figure 6). Jones et al. reported the biodistribution of m-THPC, which was administered intravenously to BDXI rats implanted with a fibrosarcoma (LSBD1).36 Their results show that 24 h after injection, the tumor-to-skin uptake ratio of m-THPC is approximately 3. Recently, the value improved to approximately 6 using m-THPC conjugated to polyethylene glycol.37 Rong et al. studied the biodistribution of HPPH in mice bearing 4T1 breast cancer cells that were introduced through intraperitoneal injection, which is very similar to the conditions described here. Rong et al. used a 64Cu complex of HPPH to quantify its biodistribution and reported a tumor-to-skin uptake ratio of approximately 2.38 Therefore, the very low biodistribution of 7 in skin is a unique feature that reduces skin sensitivity. In vivo PDT study. Mice bearing 4T1 breast cancer cells were intraperitoneally injected with 7 (125 nmol·kg−1, 0.18 mg·kg−1) or mono-L-aspartyl chlorin e6 (6250 nmol·kg−1, 5.0 mg·kg−1). The mice were irradiated with a 100-W halogen lamp (40 mW·cm−2) for 20.1 min 48 or 6 h after administration of 7 or mono-L-aspartyl chlorin e6, respectively. Figure 7 shows the mice before photoirradiation and 5 days later. Figure 7 indicates that the PDT with mono-L-aspartyl chlorin e6 damaged the tumor as well as the skin around the tumor.

In contrast, the tumor was

selectively eliminated by PDT using 7, probably because of the selective accumulation of 7 in the tumor. Figure 8 shows the results of cell death assays 1 day after PDT. Figures 8a and 8b clearly indicates that the tumor treated with mono-L-aspartyl chlorin e6 was predominantly terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive, and tumors treated with 7 were mainly TUNEL-negative. Further, Figures 8c and 8d show that the specimen treated with 7 was stained with 7-aminoactinomycin D (7-ADD), in contrast to the


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specimen treated with mono-L-aspartyl chlorin e6. Therefore, the major mechanism of cell death induced by mono-L-aspartyl chlorin e6 was through apoptosis. Further, 7 induced cell death mainly through necrosis, unlike other porphyrin-based photosensitizers. Figure 9 shows bright field images of specimens stained with hematoxylin and eosin 6 days after PDT treatment with 7. Figure 9b shows that many blood vessels were stained by hematoxylin, indicating active angiogenesis in the tumor tissue. In contrast, angiogenesis was not detected in the tumor treated with 7 and alternatively appeared the in necrotic tissue (Figure 9a). These results indicate that 7 induced necrotic cell death in vivo. Most porphyrin-based photosensitizers generally induce apoptotic cell death. In contrast, there are reports of necrotic cell death caused by PDT using fullerene (C60) derivatives. Similar to the porphyrin-based photosensitizers, 1O2 is a dominant ROS generated by photosensitization using C60 in aerated organic media (e.g., the quantum yield of 1O2 is 0.96 in C6D6).39 However, the reaction pathway of photosensitization changes in an aqueous media, such as a biological environment. The oxidation potential of electronically excited 3C60 is very high (+1.14 V vs saturated calomel electrode (SCE)).40

Therefore, when C60 is added to a reducing

microenvironment, such as a biological media, the reduced anionic radical species C60•‒ could be formed upon photoirradiation. The anionic radical species can generate radical species such as superoxide (O2•−). Alverez et al. studied the in vitro photodynamic effect on Hep-2 cells of a conjugate of porphyrin and C60 under air or an argon atmosphere.41 They found that apoptotic cell death was dominant in the air, whereas necrotic cell death increased compared with apoptosis in the argon atmosphere. Therefore, the mode of cell death may depend on the properties of the microenvironment, such as hypoxemia.

The type II photoreaction that

generates 1O2 requires aeration such as that of in vitro cultures propagated in air or perfused


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tissues in vivo. Competing reactions such as electron transfer that generate radical species may occur in hypoxemic conditions. For example, Mroz et al. studied PDT of mice with colon adenocarcinoma using i.p. injection of C60 derivatives and found that necrotic cell death was dominant compared with apoptosis, which was determined using the TUNEL assay.42 The reduction potential of electronically excited TFPP was estimated as approximately +1.15 V vs SCE.43-45 The value is comparable to that of the electronically excited 3C60 and much higher than that of the electronically excited state of 5,10,15,20-tetraphenyporphyrin estimated as approximately +0.65 V vs SCE.46,47

Therefore, it is reasonable to conclude that the

photodynamic effect of TFPP derivatives resemble that of C60, particularly in an in vivo microenvironment with relatively lower oxygen concentrations. Figure 10 shows the tumor growth curves from 0 to 7 days after PDT with 7 or mono-Laspartyl chlorin e6 at various doses. The tumor almost disappeared when injected with 6250 nmol·kg−1 (5.0 mg·kg−1) of mono-L-aspartyl chlorin e6. However, 125–625 nmol·kg−1 (0.18– 0.50 mg·kg−1) of mono-L-aspartyl chlorin e6 did not eliminate the tumor. In contrast, the tumor treated with 7 was nearly eliminated using 31.25 nmol·kg−1 (0.045 mg·kg−1). Therefore, the PDT efficacy of 7 in vivo was approximately 250-times higher than that of mono-L-aspartyl chlorin e6.

CONCLUSIONS Trans-bisthioglycosylated chlorin 7 was synthesized via 1,3-dipolar cycloaddition of azomethine ylide to the precursor trans-bisthioglycosylated porphyrin 3. Upon photoirradiation, 7 was a slightly better 1O2 generator than 3 and 8. However, the efficiency of hydroxyl radical generation by 7 was similar to that of 8 and less than that of 3. The in vitro photocytotoxicities


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to HeLa cells increased in the order of 8 < 3 < 7. This was likely to be caused by enhanced cellular uptake of the trans-bisthioglycosylated structure (i.e., 7, 3 > 8) and the greater light absorption of the chlorin ring (i.e., 7, 8 > 3).

The excellent photocytotoxicity of 7 was

established using various malignant cell lines, such as weakly metastatic B16F1 melanoma cells, metastatic 4T1 breast cancer cells, and three human glioblastoma cell lines (U87, U251, and T98G). In vivo PDT efficacy of 7 was evaluated using mice bearing 4T1 breast cancer cells via intraperitoneal injection. Biodistribution analysis indicated that the slow clearance of 7 and the tumor-to-skin ratio of 7 concentrations reached 60, 12 h after injection. The high tumor-to-skin ratio was very advantageous for reducing photosensitivity. Upon photoirradiation of the tumor site, 7 induced a significant decrease of the tumor volume even at a very low dose (31.25 nmol·kg−1, 0.045 mg·kg−1) compared with that of the second-generation photosensitizer, monoL-aspartyl

chlorin e6. The TUNEL assay indicated that necrosis was the dominant mode of cell

death caused by 7, which is similar to that of C60. This may be explained by the high oxidation potential of the excited state of fluorinated porphyrin derivatives, which is comparable to that of C60. Therefore, trans-bisthioglycosylated chlorin 7 is a powerful PDT photosensitizer in an aerated microenvironment in vitro and in a hypoxemic microenvironment in vivo.

EXPERIMENTAL SECTION Materials. All chemicals were of analytical grade. Tetraphenylporphyrin tetrasulfonic acid (TPPS)

was

purchased

from

Sigma-Aldrich

Japan

Corporation

(Tokyo,

Japan).

Hematoporphyrin (HP) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Mono-L-aspartyl chlorin e6 (9) (purity > 95%) was kindly provided by Meiji Seika Pharma Co., Ltd (Tokyo, Japan). Hydroxyphenyl fluorescein (HPF) was purchased from Sekisui


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Medical Co. Ltd. (Tokyo, Japan). MitoTraker Green FM was purchased from Molecular Probes (Eugene, OR, USA). GFP-certified apoptosis/necrosis detection system was purchased from Funakoshi Co. (Tokyo, Japan). In Situ Cell Death Detection Kit, AP was purchased from Roche Diagnostics K.K. (Tokyo, Japan). Glycoconjugated photosensitizers 328 (purity > 96%) and 826 (purity > 97%) (Scheme 1) were prepared according to our previous papers. Stock solutions of photosensitizers were prepared by weighing the dried photosensitizers and dissolving them in dimethyl sulfoxide (DMSO, Wako Pure Chemical Industries, Ltd., Kyoto, Japan), and kept in freezer (‒30°C) until use. Instrumentation and Methods.

Preparative gel permeation chromatography (GPC) was

carried out using recycling preparative HPLC system (pump; PU-2086, Jasco Co., Tokyo, Japan, detector; RI-2031 refractive index detector, Jasco Co.) equipped with Megapak GEL 201C column (GPC, 20 mmϕ × 500 mm, Jasco Co.) using CHCl3 as an eluent. The purity of 5 was determined to be >95% on an HPLC system (pump; 1515 Isocratic HPLC system, Waters Co., MA, UV-vis detector; 2487 Dual λ Absorbance Detector, Waters Co.) equipped with silica gel column (COSMOSIL 5SL-II packed column, 4.6 mmφ × 150 mm, Nacalai tesque, Inc., Kyoto, Japan) using CH2Cl2/AcOEt (7/3, v/v) at 30°C. The purities of 3, 7 and 8 were determined to be >95% on an HPLC system (pump; PU-2080Plus, UV-vis detector; UV2075Plus, Jasco Co.) equipped silica gel column (COSMOSIL 5C18-MS-II packed column, 4.6 mm × 150 mm) using CH3CN/H2O (1/1, v/v). Elemental analyses were carried out using a Perkin-Elmer PE2400 Series II CHNS/O Analyzer (Perkin-Elmer Co., Ltd., Kanagawa, Japan).

Electron-spray

ionization time-of-flight mass spectrometry (ESI-TOF MS) was recorded in CH2Cl2 (5) and methanol (7) on a JMS-T100LC AccuTOF Mass Spectrometer (JEOL, Tokyo, Japan).

1

H and

13

C NMR spectra were recorded using JNM-AL400 (400 MHz, JEOL Ltd., Tokyo, Japan), a


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NMR-DD2 500PS (500 MHz; Agilent Technologies, CA, USA) and JNM-EC600 (600 MHz, JEOL Ltd., Tokyo, Japan) instruments.

UV-vis spectra were recorded on a V-570

spectrophotometer (Jasco Co., Ltd., Tokyo, Japan). Luminescence spectra of singlet oxygen sensitized by each photosensitizer solution was recorded using a spectrometer (Jobin Yvon SPEX fluorolog3, HORIBA, Ltd., Kyoto, Japan) equipped with a photomultiplier (NIR-PMT R5509−72, Hamamatsu Photonics K.K., Shizuoka, Japan) cooled to 193 K. Absorbance and fluorescence intensity of each well were determined using plate readers (Multiscan JX, Thermo Fisher Scientific Co., Yokohama, Japan and SPECTRA Fluor Plus, TECAN Group Ltd., Seestrasse, Switzerland). Bright field and fluorescence images of cells were taken by using a confocal laser scanning microscope (CLSM, Model LSM 510, Carl Zeiss, Jena, Germany) and a fluorescence microscope (Olympus BX50 fluorescence microscope, Olympus Co., Tokyo, Japan) equipped with a Nikon DS-2MBWc CCD camera (Nikon Co., Tokyo, Japan). Synthesis.

5,15-Bis(4-(2,3,4,6-tetra-O-acetyl-β -D-glucopyranosylthio)-2,3,5,6-

tetrafluorophenyl)-10,20-bis(pentafluorophenyl)-2,3-(methano(Nmethyl)iminomethano)chlorin 5.

Glycoconjugated porphyrin 1 (236 mg, 141 µmol), N-

methylglycine (199 mg, 20.2 mmol), paraformaldehyde (488 mg) and toluene (200 mL) were refluxed for 3 h under N2. The reaction solution was washed with distilled water (100 mL × 3), dried over Na2SO4, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (silica gel, CH2Cl2 to CH2Cl2/ethyl acetate = 1/1 (v/v)) and followed by preparative GPC to give 5 (98.1 mg, 40%) as a green powder. Purity (HPLC): 98%. ESI-TOF high resolution mass spectrometry (HRMS): m/z for C75H56N5F18O18S2 ([M+H]+) calcd 1720.27743, found 1720.27775 (error 0.32 mmu, 0.18 ppm). Anal. calcd. for C75H55O18N5F18S2 + CH3CO2CH2CH3: C, 49.81; H, 3.18; N, 3.87; Found: C, 49.63; H, 2.92; N, 3.83.


1

H NMR

16

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(600.17 MHz, CDCl3, Si(CH3)4 = 0 ppm): δ (ppm) = 8.82~8.81 (2H, m, 8,17-β-pyrroleH), 8.74~8.73 (2H, m, 8,17-β-pyrroleH), 8.56~8.49 (2H, m, 12,13-β-pyrroleH), 8.49~8.40 (2H, m, 7,18-β-pyrroleH), 5.38~5.34 (2H, m, 3-GlcH), 5.32~5.18 (6H, m, 2,3-β-pyrroleH, 2,4-GlcH), 4.35~4.26 (4H, m, 6-GlcH), 3.90~3.86 (2H, m, 5-GlcH), 3.17~3.11 (2H, m, N-CHH), 2.58~2.54 (2H, m, N-CHH), 2.22~2.21 (6H, m, N-CH3 and CH3), 2.10~2.05 (18H, m, CH3).

19

F NMR

(564.73 MHz, CDCl3, CF3CO2H = ‒76.55 ppm): δ (ppm) = ‒131.12 (4F, m, 3,5-PhFGlc), ‒133.42 (4F, m, 3,5-PhFGlc), ‒131.66 (4F, m, 3,5-PhFGlc), ‒131.82 (4F, m, 3,5-PhFGlc), ‒132.57 (4F, m, 3,5-PhFGlc), ‒132.72 (4F, m, 3,5-PhFGlc), ‒135.65 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒136.12 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒137.15 (8F, m, 2,6-PhFGlc, and 3,5PhF), ‒137.65 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒137.60 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒137.78 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒138.10 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒138.16 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒152.37 (2F, m, 4-PhF), ‒152.68 (2F, m, 4-PhF), ‒152.72 (2F, m, 4-PhF), ‒152.75 (2F, m, 4-PhF), ‒161.04 (4F, m, 2,6-PhF), ‒161.42 (4F, m, 2,6-PhF), ‒162.42 (4F, m, 2,6-PhF), ‒162.45 (4F, m, 2,6-PhF).

13

C NMR (CDCl3, 150.91 MHz, CDCl3 =

77.0 ppm): δ (ppm) = 171.11, 170.66, 170.19, 170.16, 169.45, 168.86, 168.60, 152.66, 152.34, 148.55-144.61, 142.95, 141.24, 140.33-140.01, 138.99-137.02, 136.73-136.53, 135.24, 134.94, 132.47, 132.30, 128.26-128.02, 124.21-123.85, 122.61-121.88, 116.12-115.36, 111.82-111.32, 106.75, 106.08, 97.46, 96.78, 84.51-84.35 (1-GlcC), 76.40-76.35 (5-GlcC), 73.91-73.88 (3GlcC), 70.61-70.48 (2-GlcC), 68.06-67.97 (4-GlcC), 62.88 (N-CH2-), 61.84-61.64 (6-GlcC), 53.15-53.06 (2,3-β-pyrroleC), 41.18-41.15 (N-CH3-), 20.98-20.40 (CH3). UV-vis (c = 1.25 µM, DMSO, path length = 1 cm, 25°C): λ / nm (ε × 10−4 / M−1cm−1) = 409 (17.69), 504 (1.53), 526 (sh, 0.34), 598 (0.42), 652 (4.33).


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Page 18 of 54

5,15-Bis(4-(β-D-glucopyranosylthio)-2,3,5,6-tetrafluorophenyl)-10,20bis(pentafluorophenyl)-2,3-(methano(N-methyl)iminomethano)chlorin 7.

The precursor 5

(103 mg, 59.9 µmol) was dissolved in CH2Cl2 (20 mL) and methanol (20 mL).

Sodium

methoxide was added to adjust the pH to 9. This mixture was refluxed for 15 min at 50°C and was neutralized with acetic acid. After removal of the solvent, the crude product was washed with distilled water (10 mL × 5). The crude product was purified by reverse phase column chromatography (COSMOSIL 140C18-OPN, Nacalai tesque Inc., CH3CN/H2O = 8/2, v/v) and washed with distilled water to give 7 (45.4 mg, yield 55%) as a green solid. Purity (HPLC): > 99%.

ESI-TOF HRMS: m/z for C59H40N5F18O10S2 ([M+H]+) calcd 1384.19292; found

1384.19310 (error 0.19 mmu, 0.14 ppm). Anal. calcd. for C59H39O10N5F18S2 + 4H2O: C, 48.67; H, 3.25; N, 4.81; Found: C, 48.64; H, 3.40; N, 4.31. 1H NMR (600.17 MHz, CD3OD, CHD2OD = 3.30 ppm): δ (ppm) = 8.92~8.90 (2H, m, 8,17-β-pyrroleH), 8.61~8.57 (4H, m, 7,8,12,13-βpyrroleH), 5.35~5.27 (2H, m, 2,3-β-pyrroleH), 5.08~5.04 (2H, m, 1-GlcH), 3.92~3.86 (2H, m, 6GlcH), 3.70~3.66 (2H, m, 6-GlcH), 3.47~3.41 (4H, m, 2,3-GlcH), 3.41~3.35 (4H, m, 4,5-GlcH), 3.15~3.10 (2H, m, N-CHH), 2.70~2.63 (2H, m, N-CHH), 2.12 (3H, br, CH3).

19

F NMR (564.73

MHz, CD3OD, CF3CO2H = ‒76.55 ppm): δ (ppm) = ‒133.12 (4F, m, 3,5-PhFGlc), ‒133.42 (4F, m, 3,5-PhFGlc), ‒133.76 (4F, m, 3,5-PhFGlc), ‒134.65 (4F, m, 3,5-PhFGlc), ‒137.88 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒138.12 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒138.22 (8F, m, 2,6PhFGlc, and 3,5-PhF), ‒138.33 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒138.37 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒139.95 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒139.99 (8F, m, 2,6-PhFGlc, and 3,5PhF), ‒140.11 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒140.15 (8F, m, 2,6-PhFGlc, and 3,5-PhF), ‒154.65 (2F, m, 4-PhF), ‒155.24 (2F, m, 4-PhF), ‒155.28 (2F, m, 4-PhF), ‒155.32 (2F, m, 4PhF), ‒163.00 (4F, m, 2,6-PhF), ‒163.36 (4F, m, 2,6-PhF), ‒164.58 (4F, m, 2,6-PhF), ‒164.63


18

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(4F, m, 2,6-PhF).

13

C NMR (150.91MHz, CD3OD, CD3OD = 49.0 ppm): δ (ppm) = 171.19,

170.13, 154.34, 154.16, 150.17-143.97, 143.97, 142.86, 141.72, 141.61, 140.60-138.15, 136.68, 136.43, 133.92, 133.68, 129.99-129.69, 125.83, 125.56, 122.07-121.70, 116.96-116.62, 115.25114.70, 108.52, 107.32, 99.08, 98.07, 86.81-86.45 (1-GlcC), 82.71-82.68 (5-GlcC), 79.66 (3GlcC), 75.95-75.89 (2-GlcC), 71.62-71.56 (4-GlcC), 63.89 (N-CH2-), 62.96 (6-GlcC), 54.1754.08 (2,3-β-pyrroleC), 41.31-41.28 (N-CH3). UV-vis (c = 1.25 µM, DMSO, path length = 1 cm, 25°C): λ / nm (ε × 10−4 / M−1cm−1) = 410 (15.32), 506 (1.41), 527 (sh, 0.40), 597.5 (0.50), 652 (3.87). ROS Measurements. Quantum yield of singlet oxygen generation (Φ∆). Photosensitizers 3, 8, 7 and TPPS were dissolved in D2O containing 0.1% DMSO, of which the absorbance was adjusted to be 0.4 at 432 nm (3), 385.5 nm (8), 416 nm (7) and 405 nm (TPPS), and was purged with oxygen gas for 1 min.

Luminescence spectra of singlet oxygen sensitized by each

photosensitizer solution was recorded on a spectrometer equipped with a photomultiplier cooled to 193 K. The quantum yield was calculated on the basis of the value of TPPS (Φ∆ = 0.72).48 Relative quantum yield of hydroxyl radical generation (ϕ•OH). Photosensitizers 3, 7, 8 and HP (c = 1.00 µM) and HPF (c = 2.00 µM) were dissolved in PBS containing 1% DMSO and placed into a sample tube (1 mL). Oxygen gas was introduced to the solution for 1 min prior to photoirradiation. Then, the solution was exposed to light from a 100-W halogen lamp (KBEX102A, USHIO Inc., Tokyo, Japan) through a Y-50 cutoff filter (λ >500 nm, Toshiba Co., Tokyo, Japan) at 37°C. The initial rate of fluorescence intensity increments at 535 nm was monitored by excitation at 485 nm using the plate reader (SPECTRA Fluor Plus). The rate constant was estimated from the first-order plot of fluorescence intensity increments against photoirradiation time. The relative quantum yield (ϕ•OH) was evaluated by the rate constant divided by the value


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Page 20 of 54

of the oscillator strength in the wavelength above 500 nm (f>500nm) and was normalized to the value of HP. Relative quantum yield of hydrogen peroxide (ϕH2O2). Photosensitizers 3, 7, 8 and HP (c = 1.00 µM) and NaI (c = 200 mM) were dissolved in PBS containing 1% DMSO and placed into a sample tube (1 mL).

Oxygen gas was introduced to the solution for 1 min prior to

photoirradiation. The solution was subsequently exposed to light from a 100-W halogen lamp through a Y-50 cutoff filter (λ >500 nm) at 37°C. The concentration of H2O2 was monitored by measuring the absorption at 365 nm (εmax = 2.5 × 104 M−1cm−1)49 using the UV-vis spectrophotometer.

The rate constant was estimated from the first-order plot of the H2O2

concentration to the photoirradiation time. The relative quantum yield (ϕH2O2) was evaluated by the rate constant divided by the f>500nm values and was normalized to the value for HP. In Vitro Studies. Cell culture. Human cervical cell line, HeLa (ATCC CCL-2), was obtained from Dainippon-Sumitomo Pharmaco. Ltd. (Osaka, Japan). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10% fetal calf serum (FCS, Hyclone Labratories, Inc., Logan, UT, USA). Murine melanoma cell line, B16F1 (ATCC CRL-6323), was obtained from DainipponSumitomo Pharmaco. Ltd (Osaka, Japan). Cells were grows in DMEM supplemented with 10% FCS (Hyclone Labratories). Murine breast cancer cell line, 4T1 (ATCC CRL-2539), was kindly provided by Dr. Hisataka Sabe (Department of Physiological Science, Graduate School of Medicine, Hokkaido University, Sapporo, Japan). Cells were grown in DMEM supplemented with 10% FCS (Hyclone Labratories), 2 mM L-glutamine and 1% penicillin / streptomycin (Sigma-Aldrich Co.). An MNNG-induced mutant of a rat murine RGM-1 gastric carcinoma mucosal cell line, RGK-1, were established by Matsui as a co-author of this article.50,51 Cells


20

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were grown in a 1:1 mixture of DMEM and F12 (Sigma-Aldrich Co.) supplemented with 10% FCS (Sigma-Aldrich Co.) and Antibiotic-Antimycotic (Life Technologies Japan Ltd., Tokyo, Japan). Human glioblastoma cell lines, U87 (ATCC HTB-14) and U251, were obtained from the American Type Culture Collection (Rockville, MD, USA), and cell lines, T98G (ATCC CRL1690), was obtained from Riken cell bank. U251 cells were grown in DMEM supplemented with 10% FCS (Sigma-Aldrich Co.), and U87 cells and T98G cells were grown in DMEM in a 1:1 mixture of DMEM and F12 (Sigma-Aldrich Co.) supplemented with 10% FCS (SigmaAldrich Co.). Cellular uptake. The cellular uptake of photosensitizers 3, 7, 8 by HeLa cells was examined as follows: HeLa cells (2.5 × 106 cells/well) in 1.5 mL of culture medium were plated in a 6-well plate (Nalge Nunc International, Naperville, IL, USA) and incubated for 24 h (37°C, 5% CO2). Then, 1.5 mL of 0.2 µM photosensitizer in culture medium and 2% DMSO was added to each well (two wells were used for each photosensitizer) and the plate was incubated for 24 h. The final photosensitizer concentration were 0.1 µM in the culture medium (final DMSO content was 1% in all cases). Then, the cells were washed twice with Dulbecco’s phosphate-buffered saline (PBS). The cells were lysed in 200 µL of DMSO. The absorbance of the extracts at Soret band was measured by UV-vis spectrometry. The concentration of photosensitizers was calculated on the basis of the calibration obtained for each photosensitizer in DMSO. The cellular uptake is given as the means of three replicate experiments. Photocytotoxicity Test. The photocytotoxicity of photosensitizers 3, 7, 8 and mono-L-aspartyl chlorin e6 in these cell lines (HeLa, B16F1, 4T1, RGK-1, U87, U251 and T98G cells) were examined as follows: Cells (5 × 103 cells/well) in 100 µL of DMEM containing 10% FCS were plated in a 96-well plate (Nalge Nunc International and Thermo Fisher Scientific K.K.,


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Page 22 of 54

Yokohama, Japan) and incubated for 24 h (37°C, 5% CO2). One hundred microliters of a photosensitizer in culture medium containing 2% DMSO was added to each well. The plate was then incubated at 24 h in the presence of the photosensitizers. The photosensitizer concentration was varied from 1 nM to 50000 nM in culture medium (final DMSO content was 1% in all cases). The cells were washed twice with PBS, and 100 µL of the fresh culture medium was added. The cells were exposed to light from a 100-W halogen lamp equipped with a water jacket and Y-50 cutoff filter (λ >500 nm) or R-60 cutoff filter (λ > 600 nm). The light intensity was measured by using a UV-vis power meter (ORION/TH, Ophir Optronics Ltd., Jerusalem, Israel). The irradiation time was adjusted to obtain the desired light dose of 16 J·cm−2.

The

mitochondrial activity of NADH dehydrogenase of the cells in each well was measured at 24 h after photoirradiation using WST-8 reagent (10 µL) from Cell Counting Kit-8 (Dojindo, Tokyo, Japan) according to the manufacturer’s instructions. The absorbance at 450 nm was measured using a plate reader. The percentage cell survival was calculated by normalization with respect to the value for no drug treatment. Microscopy. MitoTracker Green FM-stained HeLa cells. HeLa cells (1 × 104 cells) in 500 µL of DMEM containing 10% FCS were plated in 24-well plates, and incubated for 24 h (37°C, 5% CO2). Five hundred microliters of 1 µM photosensitizer 7 in DMEM containing 10% FCS and 2% DMSO was added to each well, and then incubation was continued for 24 h in the presence of the photosensitizer. The final concentration of photosensitizers was 0.5 µM (final DMSO content was 1%). Twenty four hours after incubation, the cells were washed twice with 500 µL of PBS, and 500 µL of 250 nM MitoTracker Green FM in Hank’s balanced salt solution (HBSS) for 30 min (37°C, 5% CO2). After incubation, the cells were washed twice with 500 µL of PBS, and PBS containing 4% paraformaldehyde. One hour after fixation, fluorescence images of cells


22

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were taken with the CLSM using an excitation wavelength of 480 nm and emission wavelength of 505~530 nm (mitochondria) and 650 nm (photosensitizer). In Vivo Studies. Female 6-week-old BALB/cByJJcl mice, with body weights ranging from 19 to 22 g were purchased from CLEA Japan, Inc. (Tokyo, Japan). Mice were kept in facilities that complied with the requirements of Nara Institute of Science and Technology Animal Experiment Committee. 4T1 breast cancer cells in 100 µL HBSS (1 × 106) were transplanted into the left rear thigh, and the mice were treated with PDT when tumor volumes approached 50– 60 mm3. Tissue Distribution.

Mono-L-aspartyl chlorin e6 and 7 were dissolved in saline and

ethanol/PEG400/H2O (2/3/5, v/v/v) solution containing 0.1% DMSO, respectively, at 6250– 31.25 nmol·kg−1, and the mice were kept in dark until sacrifice. Mice were killed 0.5 h, 6 h, and 72 h after intraperitoneal injection of the drug. The tissues were rapidly removed, washed with PBS, dried using a Kimwipe, frozen in liquid nitrogen, and stored at −30°C. Blood samples (60 µL) in ethylenediaminetetraacetic acid (EDTA) (15 µL) were added to the homogenizing medium (CH3OH/DMSO/H2O = 32/8/1, v/v/v, 120 µL) and vortexed. The plasma was separated using centrifugation at 2300×g for 10 min at 4°C. One milliliter of the homogenizing medium was added to accurately weigh 100 mg of tissue. Samples were homogenized mechanically for approximately 60 s (Digital Homogenizer, ASONE Co., Osaka, Japan) in 2 mL or 5 mL tubes. The homogenate was centrifuged twice at 2300×g for 10 min at 4°C. The supernatant was removed in another tube and stored at 4°C in the dark. The supernatants were diluted with HPLC eluent (CH3CN/H2O = 7/3, v/v for 7 and ethyl acetate/CH3OH = 1/1, v/v for mono-Laspartyl chlorin e6) and analyzed using HPLC. The HPLC conditions for 7 were as follows: eluent, CH3CN/H2O = 1/1, v/v; column, COSMOSIL 5C18-MS-II (4.6 mm × 150 mm, Nacalai


23


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Page 24 of 54

Tesque, Inc., Kyoto, Japan); 40°C; flow rate, 0.75 mL·min−1; detector, fluorescence detector (λex = 404 nm, λem = 666 nm for the skin, muscle, and tumor and λem = 654 nm for other tissues). The HPLC conditions for mono-L-aspartyl chlorin e6 were as follows: eluent, ethyl acetate/CH3OH = 1/1, v/v; column, COSMOSIL 5SL-II (4.6 mm × 150 mm, Nacalai Tesque, Inc.); 30°C; flow rate, 0.5 mL·min−1; detector, fluorescence detector (λex = 400 nm and λem = 666 nm). PDT Treatment.

Mono-L-aspartyl chlorin e6 and 7 were dissolved in saline or

ethanol/PEG400/H2O (2/3/5, v/v/v) solutions containing 0.1% DMSO, at concentrations ranging from 6250–125 nmol·kg−1 (5.0–0.1 mg·kg−1) for mono-L-aspartyl chlorin e6 and 250–31.25 nmol·kg−1 (0.36–0.045 mg·kg−1) for 7, and the mice were kept in the dark. Forty-eight hours after drug administration, a 100-W halogen lamp was used to irradiate tumors at a power density of 40 mW·cm−2 for 20.1 min. Tumor growth was monitored daily by measuring tumor volume with a vernier caliper. Tumor volume was calculated using the following equation: volume = (tumor length) × (tumor width)2/2, normalized to the initial tumor volume at 0 day. Histology. The test and control mice were sacrificed 6 days after treatment with or without PDT, respectively, and their skin and tumors were resected. The samples were fixed in PBS containing 4% paraformaldehyde for 1 day at 4°C. The samples were dehydrated and embedded in paraffin. Specimens were sectioned into 7-µm slices and stained with hematoxylin and eosin, and the slides were observed using a light microscope. Apoptosis and Necrosis Assays. Cell death was investigated using slides of skin including tumor 1 day after mice were treated with PDT. Apoptosis was detected by the TUNEL assay using an In Situ Cell Death Detection Kit, AP, according to the manufacturer’s instruction (Roche Diagnostics K.K.). Slides fixed in PBS containing 4% paraformaldehyde were incubated with 15 µg·mL−1 proteinase K for 15 min at room temperature to inactivate endogenous


24

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peroxidase and washed with PBS. The slides were added to the TUNEL reaction mixture and incubated at 37°C for 60 min and then washed with PBS. Necrotic cells were detected using a GFP-certified Apoptosis/Necrosis Detection System according to the manufacturer’s instruction (Funakoshi Co.). Slides were treated with proteinase K and then added to the dual detection reagent, incubated at room temperature in the dark, and then washed with PBS. These stained slides were examined using a fluorescence microscope (Olympus BX50 equipped with a Nikon DS-2MBWc CCD camera (Nikon Corporation). Statistical Analysis. All statistical evaluations were performed using ANOVA. All values for cellular uptake, photocytotoxicity are expressed as means ± standard deviation.

ASSOCIATED CONTENT Supporting Information Available: 1H, spectra of

1

13

C and

19

F NMR spectra of 5 and 7, luminescence

O2 generated by the photosensitization using 7 and TPPS, and in vitro

photocytotoxicity of 7, 3 and mono-L-aspartyl chlorin e6 in B16F1, 4T1, RGK-1, U87, U251 and T98G cell lines. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors (S.H.) Telephone: +81-83-635-5609. Fax: +81-83-635-5469. E-mail: [email protected].

Notes


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Page 26 of 54

The authors declare no competing financial interest.

ACKNOWLEDGMENT Mono-L-aspartyl chlorin e6 was kindly provided by Meiji Seika Pharma Co., Ltd (Tokyo, Japan). This work was supported by JSPS KAKENHI Grant Numbers 23750192 and 26410189. A part of this work was conducted in NAIST, supported by Nanotechnology Platform Program (Synthesis of Molecules and Materials) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This research is supported by the Adaptable and Seamless Technology transfer Program through target-driven R&D (A-STEP: No. AS231Z00626G) from Japan.

ABBREVIATIONS USED 7-ADD, 7-Aminoactinomycin D; CTP, cell-targeting peptide; CLSM, confocal laser scanning microscope; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; EC50, the drug concentration inducing 50% cell death; EDTA, ethylenediaminetetraacetic acid; ESITOF, electron-spray ionization time-of-flight; FCS, fetal calf serum; FR, folate receptor; GPC, gel permeation chromatography; HBSS, Hank’s balanced salt solution; HP, hematoporphyrin; HPF,

hydroxyphenyl

fluorescein;

HPD,

hematoporphyrin

derivatives;

HPPH,

2-(1’-

hexyloxyethyl)-2-devinylpyropheophorbide-a; HRMS, high resolution mass spectrometry; mTHPC, 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin; PBS, Dulbecco’s phosphate-buffered saline; PDT, photodynamic therapy; ROS, reactive oxygen species; SCE, saturated calomel


26

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


electrode; TFPP, 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin; TPPS, tetraphenylporphyrin tetrasulfonic acid; TPP, 5,10,15,20-tetraphenylporphyrin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; Φ∆, quantum yield of singlet oxygen; ϕH2O2, relative quantum yield of hydrogen peroxide; ϕ·OH, relative quantum yield of hydroxyl radical.


27


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electronic spectra and four-orbital energies of free-base, zinc, copper, and palladium tetrakis(perfluoropheny1)porphyrins. Inorg. Chem. 1980, 19, 386‒391. (46) The same calculation was carried out for the electronically excied TPP with the Ered value of ‒1.23 V vs Ag/AgCl and the λmax value of Q(0,0) of 646 nm (ref. 47). (47) Bhyrappa, P.; Sankar, M.; Varghese, B. Mixed substituted porphyrins: structural and electrochemical redox properties. Inorg. Chem. 2006, 45, 4136‒4149. (48) Redmond, R. W.; Gamlin, J. N. A compilation of singlet oxygen yields from biologically relevant molecules. Photochem. Photobiol. 1999, 70, 391–475. (49) Kazantseva N. N.; Ernepesova A.; Khodjamamedov A.; Geldyev O. A.; Krumgalz B. S. Spectrophotometric analysis of iodide oxidation by chlorine in highly mineralized solutions. Anal. Chim. Acta. 2002, 456, 105–119. (50) Kobayashi I.; Kawano S.; Tsuji S.; Matsui H.; Nakama A.; Sawaoka H.; Masuda E.; Takei Y.; Nagano K.; Fusamoto H.; Ohno T.; Fukutomi H.; Kamada T. RGM1, A cell line derived from normal gastric mucosa of rat. In Vitro Cell Dev. Biol. Anim. 1996, 32, 259–261. (51) Shimokawa O.; Matsui H.; Nagano Y.; Kaneko T.; Shibahara T.; Nakahara A.; Hyodo I.; Yanaka A.; Majima H. J.; Nakamura Y.; Matsuzaki Y. Neoplastic transformation and induction of H+,K+-adenosine triphosphatase by N-methyl-N’-nitro-N-nitrosoguanidine in the gastric epithelial

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cell

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2008,

44,

26–30.

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Table 1 UV-vis Spectral Data of 3, 7, 8, and Mono-L-aspartyl Chlorin e6 (9) in PBS containing 1% DMSO at 25ºCa λmax / nm (εmax × 10−4 / M−1cm−1) Soret band 391 (3.20)c 7

424 (4.02) 391 (4.34)c

3

432 (6.53) 398 (8.22)c

8 9 a

408 (10.67) 400 (14.01)

Q bands 508 (1.16)

534 (0.62)c

599 (0.18)

656 (1.73)

512 (1.72)

544 (0.62)c

586 (0.65)

638 (0.15)

507 (0.93)

534 (0.33)c

597 (0.23)

648 (1.80)

502 (0.95)

538 (0.27)c

599 (0.38)

654 (3.06)

b

The concentrations of photosensitizers were 1.25 µM. Oscillator strength in the wavelength range above 600 nm estimated as 4.32 × 10-9∫ε(ν)dν. c Shoulder peak.


36

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2 Oscillator Strength (f)a, Quantum Yield of Singlet Oxygen (Φ∆)b and Relative Quantum Yield of Hydrogen Peroxide (ϕH2O2)c and Hydroxyl Radical (ϕ•OH)c of 7, 3, 8, TPPS, and HP.

f>600nm

f>500nm

7

0.061

0.15

0.25

0.18

1.2

3

0.017

0.14

0.16d

0.8d

7.7d

8

0.054

0.11

0.17e

1.7

1.1

0.11

0.72f

0.061

n.d.

TPPS HP a

Φ∆

ϕH2O2

n.d. 1

ϕ•OH

n.d. 1

b

Solv., PBS containing 1% DMSO. Solv., O2-saturated D2O containing 0.1% DMSO. c Solv., O2-saturated PBS containing 1% DMSO. d These values were cited from ref. 30. e The value was cited from ref. 29. f The value was cited from ref. 48.


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Table 3 Photocytotoxicity of Photosensitizers 7 and 3 in Various Malignant Cell Linesa EC50 (nM) 7

3

HeLa

0.5-1

10-50

B16F1

0.5-1

1-5

4T1

0.5-1

10-50

RGK-1

1-10

10-100

U87

0.1-0.5

10-100

U251

0.5-1

10-100

0.5-1

10-100

T98G a

The light dose was 16 J·cm >600 nm).

−2

from a 100-W halogen lamp equipped with a R-60 cutoff filter (λ


38

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 4 The Concentrations (ng/g organs) of 7 and Mono-L-aspartyl chlorin e6 (9) at Different Time Intervals after Intraperitoneal Injectiona

7 time (h)

6

12

24

48

72

brain

NDb

NDb

NDb

NDb

NDb

lung

70 ± 20

30 ± 10

29 ± 14

11 ± 3

7±7

40 ± 20

heart

24 ± 19

6±3

2.1 ± 1.5

3.3 ± 1.4

10 ± 9

stomach

48 ± 14

20 ± 10

31 ± 8

6±2

7±4

58 ± 14

liver

109 ± 16

60 ± 11

60 ± 20

9±7

14 ± 18

90 ± 50

small intestine

90 ± 40

80 ± 50

39 ± 8

9.5 ± 1.0

large intestine

80 ± 20

41 ± 3

60 ± 30

kidney

38 ± 7

25 ± 7

spleen

90 ± 60

bladder

90 ± 80

6.1 ± 1.5

6 10.2 ± 0.6

9.8 ± 0.2

90 ± 10

17 ± 5

7±5

60 ± 20

18 ± 11

5±4

13 ± 18

30 ± 9

55 ± 18

58 ± 17

14 ± 16

8±9

78 ± 19

95 ± 18

77 ± 8

3±5

8±9

14 ± 6

2.2 ± 0.6

4±3

1.2 ± 0.8

NDb

NDb

12 ± 3

skin

5±5

0c

0c

NDb

NDb

12 ± 3

tumor

5±2

4±3

muscle

a

9

3.0 ± 1.0 −1

1.4 ± 0.6

1.8 ± 1.0

34 ± 18

−1

Photosnsitizer 7 dose was 125 nmol·kg (0.18 mg·kg ) in ethanol/PEG400/H2O (2/3/5 (v/v/v)) solution containing 0.1% DMSO; mono-L-aspartyl chlorin e6 dose was 6250 nmol·kg−1 (5.0 mg·kg−1) in saline; values are the mean ± standard deviation of three animals per time point. b ND, not done. c less than 0.1.


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Page 40 of 54

Chart 1 Structure of mono-L-aspartyl chlorin e6 (9)


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Chart 2 First Generation Glycoconjugated Tetraphenylporphyrin and Optimized One 7


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Scheme 1 Syntheses of Photosensitizer 7 and its Analogues 3 (Porphyrin Analogue) and 8 (Fully Glycosylated Analogue)


42

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Figure 1 UV-vis spectra of photosensitizers 3, 7, 8 and mono-L-aspartyl chlorin e6 in PBS containing 1% DMSO at 25ºC. The concentrations of each photosensitizers were 1.25 µM.


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Figure 2 Plots of cell survival rate of HeLa cells (%) as a function of the concentration of 7, 8, 3, and mono-L-aspartyl chlorin e6. The light dose was 16 J·cm−2 from a 100-W halogen lamp equipped with a Y-50 cutoff filter (λ >500 nm) (a) or a R-60 cutoff filter (λ >600 nm). Values are the mean ± standard deviation of six replicate experiments.


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Figure 3 Relative uptake amount of 7, 3, and 8 in HeLa cells. The initial concentration of each photosensitizer was 0.1 µM.

Values are the mean ± standard deviation of three replicate

experiments. ***Significant difference (p < 0.001).


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Page 46 of 54

Figure 4 Confocal fluorescence images of HeLa cells incubated for 24 h with 7 at a final concentration of 0.5 µM and co-incubated for 30 min with 250 nM MitoTracker Green FM. Three panels show red fluorescence image (λex = 488 nm, λem = 650 nm) of 7 (a), green fluorescence image (λex = 488 nm, λem = 505‒530 nm) of MitoTracker Green FM staining mitochondria (b) and merged image (c). Scale bars: 50 µm.


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Figure 5 The concentrations of 7 and mono-L-aspartyl chlorin e6 in plasma at different times after i.p. injection of 125 nmol·kg−1 (0.18 mg·kg−1) in ethanol/PEG400/H2O (2/3/5 (v/v/v)) solution containing 0.1% DMSO of 7 and 6250 nmol·kg−1 (5.0 mg·kg−1) in saline of mono-Laspartyl chlorin e6. Values are the mean ± standard deviation of three animals per time point.


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Figure 6 The concentration of 7 (a) and mono-L-aspartyl chlorin e6 (b) in muscle, skin and tumor at different times after i.p. injection of 125 nmol·kg−1 (0.18 mg·kg−1) in ethanol/PEG400/H2O (2/3/5 (v/v/v)) solution containing 0.1% DMSO of 7 and 6250 nmol·kg−1 (5.0 mg·kg−1) in saline of mono-L-aspartyl chlorin e6. Values are the mean ± standard deviation of three animals per time point. λex = 404 nm, λem = 654 nm (7); λex = 400 nm, λem = 666 nm (mono-L-aspartyl chlorin e6).


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Figure 7 Female BALB/c mice baring 4T1 breast cancer cells before photoirradiation (a) and 5 days after PDT treatment (b) from 6 h after administration of mono-L-aspartyl chlorin e6 (6250 nmol·kg−1, 5.0 mg·kg−1), and 5 days after PDT treatment (c) from 48 h after administration of 7 (125 nmol·kg−1, 0.18 mg·kg−1).


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Figure 8 Photomicrographs of skin including tumor of female BLAB/c mice baring 4T1 breast cancer cells at 1 day after PDT treatment with 7 (a and c) and mono-L-aspartyl chlorin e6 (b and d). Left panels (a and b) show the photomicrographs labeled for apoptosis using the TUNEL assay and right panels (c and d) show the photomicrographs labeled for necrosis using the GFPcertified apoptosis/necrosis detection kit.

Drug dose were 6250 (5.0 mg·kg−1) and 31.25

nmol·kg−1 (0.045 mg·kg−1) for mono-L-aspartyl chlorin e6 and 7, respectively. Light dose was 48 J·cm-2 provided by 100-W halogen lamp (λ >500 nm).


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Figure 9 Microscopic image obtained after hematoxylin/eosin (H/E) staining of skin including tumor of female BALB/c mice baring 4T1 breast cancer cells at 6 days after PDT treatment (a) and that of the untreated control (b). The PDT treatment was carried out by photoirradiation with 100-W halogen lamp (λ > 500 nm, light dose was 48 J·cm−2) at 48 h after administration of 7 (drug dose was 31.25 nmol·kg−1 (0.045 mg·kg−1)).


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Figure 10 Reduction in female BALB/c mice baring 4T1 breast cancer cells as a function of the time after the PDT treatment with 7 (a) and mono-L-aspartyl chlorin e6 (b). The tumor volume was normalized to the initial volume. The dose of 7 were ranged from 250 to 62.5 nmol·kg−1 (from 0.36 to 0.045 mg·kg−1) and administrated as ethanol/PEG400/H2O (2/3/5, v/v/v) solution containing 0.1% DMSO. The dose of mono-L-aspartyl chlorin e6 were ranged from 6250 to 125 nmol·kg−1 (from 5.0 to 0.10 mg·kg−1) and administrated was saline. The time for accumulation of photosensitizers took 48 h for 7 and 6 h for mono-L-aspartyl chlorin e6. Values are the mean


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± standard deviation of three animals per time point. Light dose was 48 J·cm−2 provided by 100W halogen lamp (λ >500 nm).


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Table of Contents


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Synthesis, Photophysical Properties, and Biological Evaluation of

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